This invention relates generally to the silage process and to microorganisms and use of the same in treating animal feed and silage to enhance aerobic stability of the same.
The ensiling process is a method of moist forage preservation and is used all over the world. Silage accounts for more than 200 million tons of dry matter stored annually in Western Europe and the United States alone. The concept involves natural fermentation, where lactic acid bacteria ferment water soluble carbohydrates to form organic acids under anaerobic conditions. This causes a decrease in pH, which then inhibits detrimental microbes so that the moist forage is preserved. The process can be characterized by four different phases.
Upon sealing in the storage unit, the first phase is aerobic, when oxygen is still present between plant particles and the pH is 6.0 to 6.5. These conditions allow for continued plant respiration, protease activity and activity of aerobic and facultative aerobic microorganisms.
The second phase is fermentation, which lasts several days to several weeks after the silage becomes anaerobic. Lactic acid bacteria grow and become the primary microbial population thereby producing lactic and other organic acids, decreasing the pH to 3.8 to 5.0.
The third phase is stable with few changes occurring in the characteristics of the forage so long as air is prevented from entering the storage unit.
The final phase is feedout when the silage is ultimately unloaded and exposed to air. This results in reactivation of aerobic microorganisms, primarily yeast, molds, bacilli and acetic acid bacteria which can cause spoilage.
Aerobic instability is the primary problem in silage production. Even before storage units are open for feedout, silage can be exposed to oxygen because of management problems (i.e., poor packing or sealing). Under these types of aerobic conditions, rapid growth of yeast and mold cause silage to and spoil, decreasing its nutritional value.
Aerobic instability can be a problem even in inoculated silage that has undergone what would traditionally be considered a xe2x80x9cgoodxe2x80x9d fermentation phase, namely a rapid pH drop, and a low terminal pH. The yeast which contribute to instability in these conditions may be those which are tolerant of acid conditions and can metabolize the lactic acid produced by lactic acid bacteria during fermentation.
Management techniques that can be used to help prevent this condition involve using care to pack the silage well during the ensiling process and, also, using care in removing silage for feeding to minimize the aeration of the remaining silage.
The susceptibility of silage to aerobic deterioration is determined by physical, chemical, and microbiological factors. Management (compaction, unloading rates) largely effects the movement of oxygen into silage. During feedout, air can penetrate 1 to 2 m behind the silage face so that exposure to oxygen is prolonged. Fermentation acids and pH inhibit the rate of microbial growth but spoilage rates are affected also by microbial numbers and the rate of aerobic microbial growth on available substrates.
It is possible to use both chemical and biological additives in making silage to promote adequate fermentation patterns especially under sub-optimal conditions. Biological additives comprise bacterial inoculants and enzymes. Bacterial inoculants have advantages over chemical additives because they are safe, easy to use, non-corrosive to farm machinery, they do not pollute the environment and are regarded as natural products.
Lactic acid bacteria (LAB) are present as part of the normal microflora on growing plants. LAB can be classified as one of two types depending upon their primary metabolic end products; homofermentative which produce only lactic acid from the metabolism of glucose and hetrofermentative which produce lactic acid, ethanol, acetate and CO2. The occurrences of these types are quite variable in both type and number, crop to crop and location to location. There appears to be some dependence upon the environmental conditions but in general it appears that the ensiling process is dominated by homfermentative LAB.
Nilson (Arch Microbiol. (1956) 24: 396-411) found that the predominant LAB in silage are Streprococci and Lactobacilli with L. plantarum being the most frequent species. Gibson et al, J. Gen. Micro. (1958) 19: 112-129) reported that L. plantarum and L. acidophilis were the dominant component of the homofermentative flora. Beck (Landwirtschaftliche Forschung. (1972) 27: 55-63) showed that even in grass silage where the epiphyte population was dominated by heterofermentative LAB, by day four of the ensiling process 85% of the organisms were homofermentative. Langston et al. (USDA Technical Bullitin No. 1187 (1958)) has shown that the 69% of the isolates in mature silage were homofermentative. A shift is sometimes noted toward homofermentative LAB in mature silage owing to their own tolerance to low pH and high acetate concentrations. Szigeti (Acta Almentaria. (1979) 8:25-40) found that the LAB flora at extremely low pH consisted mainly of L. plantarum and L. brevis. Grazia and Suzzi (J. Appl. Bacteriol. (1984) 56: 373-379) have shown that a strong sensitivity to pH 3.6 was observed among the herofermentative LAB. The lack of pH tolerance coupled with the predominance of homofermentative LAB early in ensiling would suggest against inoculation of silage with a combination of homofermentative and homofermentative LAB.
The ensiling process is a complex one and involves interactions of numerous different chemical and microbiological processes. Further, different silages and different methods of ensiling present a variety of different needs. As can be seen a need exists in the art for further improvement in compositions and methods to improve the aerobic stability of silage. The present invention provides novel strains of L. buchneri and superior combinations of homofermenters and heterofermenters for use as silage inoculants.
The present invention provides surprisingly effective, isolated and purified combinations of the homofermentive lactic acid bacteria L. plantarnu with the heterofermentive lactic acid bacteria L. buchneri or L. brevis for use as silage inoculants. The silage inoculants provided herein provide sufficiently low pH to assure adequate preservation of ensiled forage while retaining the aerobic stability enhancement imparted by the heterofermentive bacteria. The silage inoculant has a ratio of viable homofermentive bacteria to heterofermentive bacteria of about 1:5 to about 1:15. In optional embodiments the preferred ratio is 1:10. In additional embodiments L. buchneri, such as strains LN1391, LN4637, or LN4750, is provided. In some embodiments the silage inoculant will comprise a viable culture of Enterococcus faecium. 
The present invention also provides methods of treating animal feed or silage with the silage inoculant of the present invention, as well as the treated animal feed or silage itself. Often, the animal feed or silage will be whole plant corn silage (WPCS) or high moisture corn (HWC). The present invention also provides a method of improving animal performance by feeding the inoculated animal feed or silage. Containers comprising the silage inoculant of the present invention and a carrier are also provided.
Definitions
Units, prefixes, and symbols may be denoted in their SI accepted form. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range.
As used herein, xe2x80x9cfunctional mutantxe2x80x9d means a bacterial strain directly or indirectly obtained by genetic modification of, or using, the referenced strain(s) and retaning at least 50% of the activity of a control silage using the referenced strain. The genetic modification can be achieved through any means, such as but not limited to, chemical mutagens, ionizing radiation, transposon based mutagenesis, or via conjugation, transduction, or transformation using the referenced strains as either the recipient or donor of genetic material.
As used herein, xe2x80x9cisolatedxe2x80x9d means removed from a natural source such as from uninoculated silage or other plant material.
As used herein, xe2x80x9cpurifiedxe2x80x9d means that a bacterial species or strain is substantially separated from, and enriched relative to: yeasts, molds, and/or other bacterial species or strains found in the source from which it was isolated.
As used herein, xe2x80x9canimal performancexe2x80x9d means the yield of meat, milk, eggs, offspring, or work.
In the present invention, specific bacterial species are combined in the proper ratio to provide both an adequate fermentation of silage or animal feed as well as an enhanced aerobic stability upon exposure of the silage or feed to air. The silage inoculant is an isolated and purified combination of at least one viable strain of the homofermentive lactic acid bacteria Lactobacillus plantarum and at least one viable strain of the heterofermentive lactic acid bacteria Lactobacillus buchneri or Lactobacillus brevis. In some embodiments, the silage inoculant will comprise at least 2 to 10 strains of homofermenter and/or heterofermenter. Exemplary strains of L. plantarum include at least one of LP286, LP287, LP329, LP346, LP347, or functional mutants thereof. Exemplary strains of L. buchneri include LN1391, LN4637, LN4750, or functional mutants thereof. The silage inoculant optionally comprises at least one viable strain of Enterococcus faecium, such as but not limited to, strains EF301, EF202, or functional mutants thereof. The number of viable homofermentive bacteria and heterofermentive bacteria in the inoculant are present in a ratio of from about 1:5 to about 1:15. In some embodiments the ratio is about: 1:6 to 1:14, 1:7 to 1:13, 1:8 to 1:12, 1:9 to 1:11, or 1:10.
The compositions which are used in the method of the invention may be in either liquid or dry form and may contain additional bacterial strains. In solid treatment forms, the composition may comprise the mixed bacterial culture together with a carrier. The carrier may be in the nature of an aqueous or nonaqueous liquid or a solid. In solid forms, the composition may contain solid carriers or physical extenders. Examples of such solid carriers, solid diluents or physical extenders include malto-dextrin, starches, calcium carbonate, cellulose, whey, ground corn cobs, and silicon dioxide. In short, the carrier may be organic or an inorganic physical extender. The solid composition can be applied directly to the forage in the form of a light powder dusting, or if it is disbursed in a liquid carrier it can successfully be sprayed on the forage.
Typical application rates for treating animal feed or silage according to this invention is about 104 to 106 viable homofermentive organisms/gm, preferably about 105 to 106 viable homofermentive organisms/gm.
Those of ordinary skill in the art will know of other suitable carriers and dosage forms, or will be able to ascertain such, using routine experimentation. Further, the administration of the various compositions can be carried out using standard techniques common to those of ordinary skill in the art.
Materials that are suitable for ensiling or storage, according to the methods of the invention, are any which are susceptible to aerobic spoilage. The material will usually contain at least 25% by weight dry matter. Such materials include rye or traditional grass, maize, including high moisture corn (HMC), whole plant corn (WPC), lucerne, wheat, legumes, sorghum, sunflower, barley or other whole crop cereals. The silage may be in bales (a form particularly susceptible to aerobic spoilage), oxygen limiting bags, bunkers, upright stave silos, oxygen limiting silos, bags, piles or any other form of storage which may be susceptible to aerobic spoilage. Alternatively, the invention may be used with any susceptible animal feed, whether solid or liquid, e.g. for pigs, poultry or ruminants.
Deposits
A deposit of the following microorganisms has been made with the American Type Culture Collection (ATCC), Rockville, Md., 20852: LP286 (ATCC Accession No. 53187), LP287 (ATCC Accession No. 55058), LP329 (ATCC Accession No. 55942), LP346 (ATCC Accession No. 55943), LP347 (ATCC Accession No. 55944), EF301 (ATCC Accession No. (55593), EF202 (ATCC Accession No. 53519), LN1391 (ATCC Accession No. PTA-2493), LN4637 (ATCC Accession No. PTA-2494), LN4750 (ATCC Accession No. PTA-2495). Deposit dates are as follows: LN 1391, LN 4637, and LN 4750 on Sep. 21, 2000, LP286 on May 11, 1985; LP287 on Jun. 5, 1990; LP329, LP346, and LP347 on Mar. 5, 1997; EF301 on Jun. 21, 1994, and EF202 on Jul. 23, 1986. The microorganisms deposited with the ATCC were taken from the same deposit maintained at Pioneer Hi-Bred International, Inc (Des Moines, Iowa.). Applicant(s) will meet all the requirements of 37 C.F.R. xc2xa71.801-1.809, including providing an indication of the viability of the sample when the deposit is made. These deposits will be maintained without restriction in the ATCC Depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it ever becomes nonviable during that period.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.